Disorders of the internal anal sphincter (IAS) underlie many clinical abnormalities, such as fecal incontinence and constipation, and emphasize its role in continence and defecation. Disorders of the IAS occur more frequently in the rapidly expanding population of the elderly, and thus give increased urgency to understanding its function. Therefore, the objectives of this research proposal are: 1) to investigate intracellular mechanisms that regulate the basal tone of the LAS, and 2) to determine the role of cellular and biochemical elements in the nonadrenergic noncholinergic (NANC)-mediated relaxation of the IAS. Relations between electrical and mechanical activities will be determined by simultaneous recordings in IAS isolated from the external anal sphincter (EAS), in vivo and in vitro. Correlations between intracellular biochemistry and contraction-relaxation of the IAS will be obtained by measurements of changes in membrane potentials, flux in free intracellular Ca2- turnover of Pt, G-proteins, activities of protein kinase C, myosin light chain kinase (MLCK) and MLC-phosphatase, and the state of phosphorylation of myosin light chain (MLC20-P). In addition, we will determine cyclic nucleotides, immunocytochemical localization, direct release of the mediators, and enzymatic activities involved in their biosynthesis. Murine models with targeted disruption of a specific gene which render them deficient in either intramuscular interstitial cells of Cajal (ICC-IM; WWV), nNOS (nNOS-/-), or HO-2 (HO-2-/-) will be used to determine cellular and biochemical elements which regulate the basal tone and the nature of inhibitory neurotransmission in the LAS. Either mice or opossums will be used in almost all of the above studies, as these are well-characterized animal models for the study of the LAS. Our multidisciplinary approach to the study of the IAS will define: (1) the cellular bases and signaling pathways most critical to the regulation of the LAS tone, and (2) the nature of inhibitory neurotransmission in the IAS. Information produced by these studies will permit an understanding of the pathophysiology that underlies disorders of the LAS, and provide a rationale for development of therapies to treat disorders of the IAS.